1. Technical Field
The present invention relates to apparatus and methods for analysis of biologic fluid samples (e.g., blood) in general, and for the detection of anisotropic crystals within the samples in particular.
2. Background Information
Anisotropic (also referred to as birefringent) crystals are not usually present in liquid whole blood of healthy subjects. Whole blood that is allowed to dry will generally result in the precipitation/formation of crystals and particles that are anisotropic but have no readily detectable anisotropic features while they are in solution. Anisotropic solid substances that may be present in un-dried whole blood include: 1) phagocytosed uric acid crystals (seen in acute gout), 2) phagocytosed cholesterol crystals seen in degenerative states of intra-arterial plaque in which plaque is phagocytosed, 3) phagocytosed Charcot-Leyden crystals found in degenerating eosinophils, and 4) hemozoin, a waste product of hemoglobin digestion by hematophagous parasites infecting the vertebrate host such as those in the order Haemosporida. Other parasites that ingest hemoglobin and excrete hemozoin include Schistosomes, an important cause of morbidity throughout the world. Perhaps the medically most important cause of the presence of circulating anisotropic crystals in whole blood in humans is infection with malaria in which the crystals are hemozoin and seen in a small proportion of the red blood cells and in some cases in some monocytes and neutrophils, although the detection and identification of the other mentioned crystals is also potentially clinically useful. Hemozoin is a waste product of the parasites digestion of the red cells hemoglobin. Red blood cells infected with malarial parasites will have significant decreases in their hemoglobin content and or concentration due to the ingestion of the hemoglobin by the parasite and its eventual excretion as the waste product, hemozoin. The formation of crystalline of hemozoin renders it non-toxic to the parasite. Babesia are another genus, with over 100 species, of protozoa that are clinically important members of the order Haemosporida. Both malaria and Babesiosis are found in many species of vertebrates in addition to humans. Birds and reptiles are common hosts. Babesia, in contrast to similar appearing malarial protozoans, according to the literature, do not have hemozoin associated with their infection. This absence of hemozoin in Babesia infections can be of utility in distinguishing the morphologically similar Babesia forms from malarial infections, but obviously hemozoin detection cannot be used as a screening tool for the detection of Babesia infection, Haemoproteus is another genus of protozoa that are parasitic in birds, reptiles and amphibians. These protozoa infect both wild birds and birds in the food chain such as turkeys. The protozoa, like malaria, produce hemozoin.
Hemozoin may be found both in the red blood cells that are infected with the hematophagous parasites or in neutrophils or monocytes that have phagocytosed the infected red blood cells containing the hemozoin or have phagocytosed the free hemozoin released by lysed red blood cells or excreted by the parasite.
While the detection of hemozoin has been used to alert the physician to the possibility of the host organism being infected with a hemozoin excreting parasite, the utility of looking for hemozoin is presently severely limited by the complexity of the methods employed to detect it. Current techniques to discover the hemozoin in erythrocytes or white blood cells from patients with malaria require a relatively high-powered microscope and often require the use of dark field or polarized microscopy. Such instruments are expensive and not suitable for field use,